Sodium current is fundamental for normal and abnormal cardiac excitation and for conduction of the cardiac action potential. The cardiac voltage dependent sodium channel is the target of many currently used antiarrhythmic and cardiotonic agents. Characterization of the functional and structural properties of the cardiac "TTX-insensitive" (TTX-I) Na channel, however, has lagged behind that of "TTX-sensitive" technical difficulties which result from the complexity of heart tissue. Before the advent of single cell and patch clamp technology, the tissue geometry of the heart precluded adequate in situ voltage clamp control for detailed electrophysiological measurements. The in vitro characterization of the structure and function of TTX-I channels has been hampered because 1) cardiac TTX-1 channels are 10 - 100 times less abundant in heart that TTX-S channels are in noncardiac tissues and 2) cardiac tissue contains a significant component of contaminating TTX-S channels which complicate the contains a significant component of contaminating TTX-S channels which complicate the study of TTX-I \ channels. (Most, if not all of the TTX-S channels in the heart come from nerves present in the heart.) Despite these problems, t-tubular sarcolemmal membrane fractions from adult mammalian hearts have been prepared in this laboratory which are highly enriched in TTX-I saxitoxin (STX) binding receptors and devoid of TTX-S STX receptors. These and numerous other studies have verified that cardiac TTX-I channels have unique pharmacological, toxicological, and electrophysiological properties which are distinct form those of the TTX-S channels. The overall goal of this proposal is to determine special structural characteristics of cardiac TTX-I sodium channels responsible for their special functional characteristics. The isolation of TTX-I channels from TTX-S channels allows TTX-I channels to be assayed unambiguously for direct comparison with preparations of TTX-S channels from nerve and skeletal muscle. Specifically, Binding and competition to binding of pertinent ligands will be measured in native membrane, detergent solubilized and purified preparations. The functionality of the channels and their response to specific ligands will be assessed in parallel by electrical recording form single channels incorporated into planar lipid bilayers. Further knowledge of distinctions in the functional structure of sodium channels potentially allows for increased specificity of therepeutic drugs.